RESUMO
Macaca, Callithrix jacchus marmoset monkey, Pan troglodytes chimpanzee and human retinas were examined to define if short wavelength (S) cones share molecular markers with L&M cone or rod photoreceptors. S cones showed consistent differences in their immunohistochemical staining and expression levels compared to L&M cones for "rod" Arrestin1 (S-Antigen), "cone" Arrestin4, cone alpha transducin, and Calbindin. Our data verify a similar pattern of expression in these primate retinas and provide clues to the structural divergence of rods and S cones versus L&M cones, suggesting S cone retinal function is "intermediate" between them.
Assuntos
Opsinas dos Cones/metabolismo , Luz , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Opsinas de Bastonetes/metabolismo , Animais , Biomarcadores/metabolismo , Calbindina 1/metabolismo , Callithrix , Humanos , Macaca , Pan troglodytes , Transducina/metabolismoRESUMO
PURPOSE: We used immunocytochemistry and confocal microscopy to determine whether enzymes of the rod visual cycle were uniformly distributed in retinal pigment epithelium (RPE) cells. The localizations of these enzymes were compared to known localizations of retinoid-binding proteins and associated proteins. METHODS: Antibodies to proteins and enzymes associated with the rod visual cycle were used for fluorescence immunocytochemistry with frozen sections of albino mouse and rat retina. Images were obtained with a laser scanning confocal microscope. RESULTS: Components associated with the rod visual cycle were distributed in three distinct patterns in mouse and rat RPE. Three visual cycle enzymes (RDH5, LRAT, and RPE65) were restricted to the somata of RPE cells and were not detected within apical processes. Ezrin, an actin-binding protein, and ERM-binding phosphoprotein50/sodium-hydrogen exchanger regulatory factor1 (EBP50/NHERF1), an ezrin-binding PDZ-domain protein, were largely restricted to RPE apical processes. The fluorescence intensity over Müller cell apical processes was less intense. Cellular retinaldehyde-binding protein (CRALBP), which binds to EBP50/NHERF1, and cellular retinol-binding protein type 1 (CRBP1) were found throughout RPE cells and Müller cells. CONCLUSIONS: Visual cycle enzymes were confined to the somata of RPE cells and did not occur within the long apical processes, either in dark- or light-adapted animals. Other components previously linked to the visual cycle (EBP50/NHERF1 and ezrin) were largely confined to the apical processes, where they could be associated with release of 11-cis-retinal or uptake of all-trans-retinol. CRALBP and CRBP1 were distributed throughout the RPE cell, where they could mediate diffusion of retinoids between apical processes and somata.
Assuntos
Aciltransferases/metabolismo , Oxirredutases do Álcool/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Olho/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Actinas/metabolismo , Adaptação Ocular , Albinismo , Animais , Calreticulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Proteínas Celulares de Ligação ao Retinol/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , cis-trans-IsomerasesRESUMO
The Purkinje cell degeneration (pcd) mouse undergoes retinal photoreceptor degeneration and Purkinje cell loss. Nna1 is postulated to be the causal gene for pcd. We show that a BAC containing the Nna1 gene rescues retinal photoreceptor loss and Purkinje cell degeneration, confirming that Nna1 loss-of-function is responsible for these phenotypes. Mutation of the zinc-binding domain within the transgene destroyed its ability to rescue neuronal loss in pcd(5J) homozygous mice. In conclusion, Nna1 is required for survival of retinal photoreceptors and other neuron populations that degenerate in pcd mice. A functional zinc-binding domain is crucial for Nna1 to support neuron survival.
Assuntos
Ataxia Cerebelar/fisiopatologia , Proteínas de Ligação ao GTP/fisiologia , Células de Purkinje/patologia , Degeneração Retiniana/fisiopatologia , D-Ala-D-Ala Carboxipeptidase Tipo Serina/fisiologia , Zinco/metabolismo , Sequência de Aminoácidos , Animais , Ataxia Cerebelar/genética , Ataxia Cerebelar/patologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Genótipo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismoRESUMO
CaBP1-8 are neuronal Ca(2+)-binding proteins with similarity to calmodulin (CaM). Here we show that CaBP4 is specifically expressed in photoreceptors, where it is localized to synaptic terminals. The outer plexiform layer, which contains the photoreceptor synapses with secondary neurons, was thinner in the Cabp4(-/-) mice than in control mice. Cabp4(-/-) retinas also had ectopic synapses originating from rod bipolar and horizontal cells tha HJt extended into the outer nuclear layer. Responses of Cabp4(-/-) rod bipolars were reduced in sensitivity about 100-fold. Electroretinograms (ERGs) indicated a reduction in cone and rod synaptic function. The phenotype of Cabp4(-/-) mice shares similarities with that of incomplete congenital stationary night blindness (CSNB2) patients. CaBP4 directly associated with the C-terminal domain of the Ca(v)1.4 alpha(1)-subunit and shifted the activation of Ca(v)1.4 to hyperpolarized voltages in transfected cells. These observations indicate that CaBP4 is important for normal synaptic function, probably through regulation of Ca(2+) influx and neurotransmitter release in photoreceptor synaptic terminals.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras/metabolismo , Retina/anormalidades , Sinapses/metabolismo , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio Tipo L/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/isolamento & purificação , Linhagem Celular , Coristoma/genética , Coristoma/metabolismo , Coristoma/patologia , DNA Complementar/análise , DNA Complementar/genética , Humanos , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Neurotransmissores/metabolismo , Cegueira Noturna/genética , Cegueira Noturna/metabolismo , Células Fotorreceptoras/ultraestrutura , Retina/metabolismo , Retina/ultraestrutura , Sinapses/ultraestrutura , Transmissão Sináptica/genéticaRESUMO
PURPOSE: To determine pathways of sympathetic nerves from the orbital apex to the eyelids in human cadaver tissue using immunohistochemistry. METHODS: Human cadaver orbit tissue was sectioned and immunolabeled with a monoclonal antityrosine hydroxylase antibody. RESULTS: In the orbital apex, the nasociliary, frontal, lacrimal, and maxillary branches of the trigeminal nerve demonstrated intense staining upon entering the orbit. Immunoreactive axons from the nasociliary and frontal nerves were observed to join the extraocular motor nerves in the posterior orbit. A plexus of immunolabeled nerves was observed to accompany the ophthalmic artery as it entered the orbital apex. The ophthalmic artery and its branches throughout the orbit demonstrated staining of nerve fibers in the peripheral muscularis. The nasociliary nerve contributed sympathetic branches to the ciliary ganglion. Nerves passing through the ciliary ganglion and a few ganglion cell bodies demonstrated mild to moderate tyrosine hydroxylase reactivity. Axons within the short and long ciliary nerves demonstrated strong tyrosine hydroxylase reactivity and were observed to enter the posterior sclera and the suprachoroidal space. The lacrimal gland demonstrated mild pericapillary staining and occasional stromal nerve fibers reactive to the antityrosine hydroxylase antibody. Müller muscle and the inferior tarsal muscle possessed a strong tyrosine hydroxylase-reactive nerve supply that appeared to originate from the anterior terminal branches of the nasociliary and lacrimal nerves. CONCLUSIONS: Sympathetic nerves enter the orbit via the first and second divisions of the trigeminal nerve and a plexus of nerves surrounding the ophthalmic artery. Extraocular motor nerves receive a sympathetic nerve supply from the sensory nerves in the posterior orbit. Some ciliary ganglion cell bodies demonstrated tyrosine hydroxylase-like reactivity, suggesting a sympathetic modulatory role for the ciliary ganglion. Sympathetics innervate ocular structures via the posterior ciliary nerves. Sympathetic axons travel anteriorly in the orbit via the nasociliary and lacrimal nerves to innervate the sympathetic eyelid muscles. Sympathetic nerves also travel with the frontal branch of the ophthalmic nerve to innervate the forehead skin. The ophthalmic artery and all of its branches contain a perivascular sympathetic nerve supply that may be involved in regulation of blood flow to ocular and orbital structures.
Assuntos
Pálpebras/inervação , Vias Neurais/anatomia & histologia , Órbita/inervação , Sistema Nervoso Simpático/anatomia & histologia , Animais , Humanos , Técnicas Imunoenzimáticas , Macaca nemestrina , Artéria Oftálmica/inervação , Nervo Oftálmico/anatomia & histologia , Pele/inervação , Nervo Trigêmeo/anatomia & histologia , Tirosina 3-Mono-Oxigenase/imunologiaRESUMO
PURPOSE: To quantify changes in the lens epithelial cells and underlying lens cortex responsible for age-related cortical cataract (ARCC) in the rat. METHODS: Freshly isolated lenses were stained vitally for DNA with Hoechst 33342. Reactive oxygen species (ROS) and mitochondria were visualized and quantified by dihydrorhodamine 123 (DHR). The fluorescence was quantified using Laser Scanning Confocal Microscopy (LSCM) of vitally stained lenses. Cortical DNA was verified as such by DNAse I digestion. Cataract reflections were determined from digitalized images of light reflections taken with a low magnification light microscope, or with the LSCM. RESULTS: The anterior surface epithelia of old rat lenses were full of gaps and ragged in appearance with a decrease of over 50% in lens epithelial cell (LEC) density. The surface LECs were frequently seen to have involuted into the cortex at inappropriate sites, forming deposits full of DNA, nuclear and mitochondrial debris, and abundant ROS. These involutions frequently originated near open gaps in the surface epithelia, where they appear to have detached from the capsular membrane. Cortical cataracts in the rat lenses were seen to co-localize with these LEC involutions, as had been seen previously in mice with ARCC. CONCLUSIONS: ARCC in rats co-localized with inappropriate accumulations of nuclei, mitochondria, DNA, and expression of ROS in debris filled foci. These were the result of both involution of surface LECs into areas of cortical ARCC, and by an extension of the normal bow region deep into the anterior and posterior of cataractous lenses. These results were in complete agreement with our previous studies on ARCC in mice.
Assuntos
Envelhecimento , Catarata/metabolismo , Catarata/patologia , Córtex do Cristalino , Espécies Reativas de Oxigênio/metabolismo , Animais , Contagem de Células , Núcleo Celular/ultraestrutura , DNA/metabolismo , Células Epiteliais/patologia , Córtex do Cristalino/patologia , Microscopia Confocal , Mitocôndrias/ultraestrutura , Ratos , Ratos Endogâmicos BN , RodaminasRESUMO
Spinocerebellar ataxia (SCA) type 7 is an inherited neurodegenerative disorder caused by expansion of a polyglutamine tract within the ataxin-7 protein. To determine the molecular basis of polyglutamine neurotoxicity in this and other related disorders, we produced SCA7 transgenic mice that express ataxin-7 with 24 or 92 glutamines in all neurons of the CNS, except for Purkinje cells. Transgenic mice expressing ataxin-7 with 92 glutamines (92Q) developed a dramatic neurological phenotype presenting as a gait ataxia and culminating in premature death. Despite the absence of expression of polyglutamine-expanded ataxin-7 in Purkinje cells, we documented severe Purkinje cell degeneration in 92Q SCA7 transgenic mice. We also detected an N-terminal truncation fragment of ataxin-7 in transgenic mice and in SCA7 patient material with both anti-ataxin-7 and anti-polyglutamine specific antibodies. The appearance of truncated ataxin-7 in nuclear aggregates correlates with the onset of a disease phenotype in the SCA7 mice, suggesting that nuclear localization and proteolytic cleavage may be important features of SCA7 pathogenesis. The non-cell-autonomous nature of the Purkinje cell degeneration in our SCA7 mouse model indicates that polyglutamine-induced dysfunction in adjacent or connecting cell types contributes to the neurodegeneration.
Assuntos
Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Células de Purkinje/patologia , Degenerações Espinocerebelares/etiologia , Animais , Ataxina-7 , Núcleo Celular/patologia , Marcha Atáxica/etiologia , Marcha Atáxica/metabolismo , Marcha Atáxica/patologia , Corpos de Inclusão/patologia , Cinética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/fisiologia , Degenerações Espinocerebelares/metabolismo , Degenerações Espinocerebelares/patologiaRESUMO
PURPOSE: To characterize mechanisms of apical localization of visual cycle components in retinal pigment epithelium (RPE) by the identification of cellular retinaldehyde-binding protein (CRALBP) interaction partners. METHODS: An overlay assay was used to detect interactions of CRALBP with components of RPE microsomes. Interacting proteins were identified with two-dimensional (2D)-PAGE and liquid chromatography tandem mass spectrometry (LC MS/MS). Protein interactions were characterized by affinity chromatography, peptide competition, and expression of protein domains. Protein colocalization in mouse retina was examined using double-label immunocytochemistry and confocal microscopy. RESULTS: CRALBP bound to a 54-kDa protein in RPE microsomes, which was identified as ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 (EBP50), a PDZ domain protein, also known as sodium/hydrogen exchanger regulatory factory type 1 (NHERF-1). EBP50 and ezrin in solubilized microsomes bound to CRALBP-agarose but not to a control agarose column. CRALBP bound to both recombinant PDZ domains of EBP50 but not to the C-terminal ezrin-binding domain. In outer retina, EBP50 and ezrin were localized to RPE and Müller apical processes. CRALBP was distributed throughout both RPE and Müller cells, including their apical processes. CONCLUSION: RM proteins are multivalent linkers that connect plasma membrane proteins with the cortical actin cytoskeleton. EBP50 interacts with ERM family members through a C-terminal domain and binds targets such as CRALBP through its PDZ domains, thus contributing to an apical localization of target proteins. Our results provide a structural basis for apical localization of a retinoid-processing complex in RPE cells and offer insight into the cell biology of retinoid processing and trafficking in RPE.
Assuntos
Proteínas de Transporte/metabolismo , Fosfoproteínas/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Retinaldeído/metabolismo , Trocadores de Sódio-Hidrogênio , Animais , Bovinos , Cromatografia de Afinidade , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Humanos , Espectrometria de Massas , Camundongos , Microscopia Confocal , Microssomos/metabolismo , Ligação Proteica , Mapeamento de Interação de ProteínasRESUMO
Lack of an optimal in vitro model of human corneal epithelial (HCE) cells is a major limitation in studying normal functions and gene regulations in HCE. Moreover, availability of a multi-layered HCE culture can reduce the usage of animals in the toxicity testing of consumer products. We have developed tetracycline-responsive human papilloma virus (HPV) 16-E6/E7 transduced HCE clones showing tight regulation of proliferation and normal differentiation. Expression of HPV16-E6/E7 mRNA and HPV16-E7 and keratin K3 proteins was examined by RNase protection assay and western blotting, respectively, in presence and absence (+/-) of Dox in identified clones. Localization of cornea-specific keratin k3 in +/- of Dox was evaluated by immunocytochemistry. The response of growth factors such as hepatocyte growth factor (HGF) and epidermal growth factor to the cellular proliferation in +/- of Dox in the newly identified clones was measured by cell counting. Cellular morphology, formation of multi-layered cultures at air-liquid interface and ultrastructural features were evaluated by light and transmission electron microscopy. The physical barrier established by the newly developed clones was determined by the transepithelial permeability to sodium fluorescein and transepithelial electrical resistance assays in the airlifted-stratified cultures.